Impact Mechanism Of Pectin Complex System And Salicylic Acid On The Activity Of Polyphenol Oxidase

Polyphenol oxidase(PPO)is a typical type III copper protein which is widely distributed on thylakoids of fruit and vegetables,and plays an important role in its enzymatic browning.Therefore,the passivation of PPO activity is an effective method to realize the browning control of fruit and vegetables.Model systems such as buffers provide great convenience for the research of PPO,but the influence of food components on PPO is neglected,resulting in a big difference between the inhibition of PPO in the buffer and the actual food systems.As a common food component,pectin is widely present in fruit,vegetables and their products.It will be of great significance to explore the effects of pectin on PPO activity and enzyme inactivation.Interestingly,during the procedure of investigating the inhibitory effects of four different organic acids on PPO activity in pectin complex system,it was found that salicylic acid not only has a chemical structure similar to that of the substrate of PPO,but its acidification properties(p K_a=2.98)can also affect the inhibitory effects.However,the effects of salicylic acidification on PPO activity have not been reported so far.Consequently,this article explores the mechanism of salicylic acid inhibiting PPO activity from the aspects of acidification and binding effects.The main contents are as follows:(1)The effects of pectin complex system on PPO activity and resistance against traditional thermal and acidic passivation were investigated.Firstly,a pectin complex system was constructed by adding pectin on the basis of the buffer system,and in which the enzyme activity of PPO was measured.Secondly,PPO with pectin complex system was exposed to a thermal processing of 45-60°C and the effects of pectin on the activity and conformational stability of PPO against thermal passivation were investigated.Finally,differential inhibitory effects of four different organic acids,ascorbic acid,citric acid,ferulic acid and salicylic acid on PPO activity in the buffer and pectin complex system were investigated.Experimental results showed that pectin could inhibit PPO activity and PPO activity decreased gradually with the increase of pectin mass concentration.When the mass concentration of pectin was 10 mg/m L,PPO activity decreased by about 20%,and PPO activity did not decline further with the increase in pectin mass concentration.Thermal passivation kinetic results and fluorescence spectra showed that the presence of pectin in the complex system could reduce the sensitivity of PPO to thermal passivation and hence enhance its thermal stability.Moreover,it was found that 5 mg/m L pectin in pectin complex system could increase the inhibitory effects of ascorbic acid,citric acid,ferulic acid and salicylic acid on PPO activity by 1.5%?25.7%.(2)The mechanism of salicylic acid inhibiting PPO activity was discussed based on the aspect of acidification and binding effects.Firstly,buffer solutions with different concentrations were used to investigate the effects of salicylic acidification as well as the p H dependence of salicylic acid on PPO enzymatic activity.Secondly,the effects of salicylic acidification were eliminated by adjusting the p H of the system,and the binding effects between salicylic acid and PPO molecule as well as the inhibition types were investigated by means of inhibition kinetics and circular dichroism spectroscopy(CD).Finally,the inhibitory effects of salicylic acid on PPO activity were comprehensively analyzed under specific p H conditions,and the influence of the salicylic acidification on its binding effects with PPO molecule was discussed through molecular docking.Experimental results showed that salicylic acid is a competitive inhibitor of PPO molecules,and it inhibits PPO activity through both acidification and binding effects.Salicylic acidification was predominant in weak buffer systems,while in strong buffer systems,the contribution of binding effects increased.Hydrogen bond,hydrophobic interaction and van der Waals force are the main driving forces for the binding of PPO molecules and salicylic acid.Additionally,electrostatic interaction could be formed and more hydrogen bonds were generated under acidic p H,which enhanced the stability of the binding conformation between salicylic acid and PPO molecule.It can be known that the acidification and binding effects of salicylic acid were synergistic in the inhibition of PPO activity.In this paper,the effects of food component pectin on PPO activity,molecular conformation and enzyme passivation under traditional acidic and thermal conditions were analyzed by pectin complex system.It was confirmed that the food components were an important reason for the differential inhibitory effects of PPO in the buffer and the actual food system,so as to provide theoretical references for the application of enzymatic browning control of fruit and vegetables and their products in production practice.Then,the double action mechanism of salicylic acid inhibiting PPO activity was clarified,which provided theoretical guidance for the application of salicylic acid in controlling the enzymatic browning of fruit and vegetables.